RESUMO
PURPOSE: Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces. MATERIALS AND METHODS: hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium. RESULTS: The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins. CONCLUSION: These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.
Assuntos
Materiais Dentários/química , Células-Tronco Mesenquimais/fisiologia , Titânio/química , Condicionamento Ácido do Dente/métodos , Adulto , Fosfatase Alcalina/análise , Células da Medula Óssea/fisiologia , Proteína Morfogenética Óssea 2/análise , Adesão Celular , Diferenciação Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Colágeno/biossíntese , Colágeno Tipo I/análise , Subunidade alfa 1 de Fator de Ligação ao Core/análise , DNA/biossíntese , Humanos , Sialoproteína de Ligação à Integrina , Microscopia Eletrônica de Varredura , Células-Tronco Multipotentes/fisiologia , Osteoblastos/citologia , Osteocalcina/análise , Osteopontina/análise , Prolina/metabolismo , Sialoglicoproteínas/análise , Fator de Transcrição Sp7 , Propriedades de Superfície , Timidina/metabolismo , Fatores de Transcrição/análiseRESUMO
PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.
Assuntos
Osteoblastos/metabolismo , Titânio , Análise de Variância , Diferenciação Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Expressão Gênica , Humanos , Sialoproteína de Ligação à Integrina , Mandíbula/citologia , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Osteoblastos/ultraestrutura , Osteonectina/biossíntese , Osteopontina/biossíntese , RNA/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/biossíntese , Propriedades de Superfície , Fator de Crescimento Transformador beta2/biossínteseRESUMO
This study evaluated the effects of commercially available antiseptic mouthrinses on human gingival fibroblast and keratinocyte behaviour and metabolism. Three mouthrinses containing essential oil (EO), chlorhexidine (CHX) and amine fluoride/stannous fluoride (AFSF), were tested in an in vitro study. Human gingival fibroblasts and keratinocytes were washed with 10% or 30% concentration of the commercial mouthrinses and their effects on cell adhesion and proliferation were investigated as well as the specific gene expression of markers involved in oral mucosa metabolism. As markers of cell metabolism, type I and IV collagens, laminin, fibronectin, fibromodulin and integrins were studied with real-time PCR. Moreover, interleukin-1 secretion, one of the major pro-inflammatory cytokines, was evaluated. The results showed that CHX significantly reduced fibroblast and keratinocyte substrate adhesion capacities and CHX and EO inhibited cell proliferation better than AFSF rinse. The gene expression of several matrix components and cell adhesion receptors was downregulated in cells washed with CHX and EO compared with those washed with AFSF rinse. In conclusion, the AFSF mouthrinse does not induce or induces to a lesser extent the onset of irritation and/or cytotoxicity than CHX or EO. These findings and those of future studies will enable us to gain further insight into the clinical significance and effects of commercial mouthrinses. Pending further investigations, clinicians should be aware of the potentially adverse effects of mouthrinses and warn their patients against making improper use of these products.
Assuntos
Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Queratinócitos/efeitos dos fármacos , Antissépticos Bucais/toxicidade , Anti-Infecciosos Locais/toxicidade , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Clorexidina/toxicidade , Colágeno Tipo I/genética , Colágeno Tipo IV/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibromodulina/genética , Fibronectinas/genética , Fluoretos Tópicos/toxicidade , Expressão Gênica/efeitos dos fármacos , Humanos , Integrinas/genética , Interleucina-1 , Queratinócitos/metabolismo , Laminina/genética , Óleos Voláteis/toxicidade , RNA Mensageiro/metabolismo , Fluoretos de Estanho/toxicidadeRESUMO
Cyclosporin A is a powerful immunosuppressive drug that has had a major impact on transplant therapy. It apparently links to different enzymatic pathways, and affects multiple enzymatic systems. Transforming growth factor beta induces the deposition of glycosaminoglycans, proteoglycans, and collagen fibers in the extracellular matrix. The aim of this study of normal and hypertrophic human gingival fibroblast cultures was to evaluate the cytoskeletal and extracellular changes in glycosaminoglycan secretion due to the presence of cyclosporin A and transforming growth factor beta. The results showed that there is an increase in total and individual classes of extracellular glycosaminoglycans in the presence of cyclosporin A and transforming growth factor beta, but the action of the latter was significantly greater. Immunohistochemical analysis of the cytoskeleton did not reveal any morphological differences between treated and control cells. Our data suggest that the biochemical changes in the extracellular matrix are caused more by cytokine, and that cyclosporin A does not induce any morphological changes in fibroblast cultures derived from hypertrophic and normal gingiva.
Assuntos
Ciclosporina/farmacologia , Citoesqueleto/metabolismo , Gengiva/metabolismo , Glucosamina/metabolismo , Glicosaminoglicanos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Células Cultivadas , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gengiva/citologia , Gengiva/efeitos dos fármacos , Humanos , Imunossupressores/farmacologiaRESUMO
Crosslinking of collagen biomaterials increases their resistance to degradation in vivo. Glutaraldehyde (GA) is normally used to crosslink collagen biomaterial, but is often cytotoxic. Diphenylphosphoryl azide (DPPA) has recently been proposed as reagent, but little is known about its effects on cell behavior. In this study, we determined which collagen membrane was the most biocompatible: Paroguide which is crosslinked with DPPA and contains chondroitin sulfate; Opocrin which is crosslinked with DPPA; Biomed Extend which is crosslinked with GA; and Bio-Gide which is left untreated. Cell proliferation and extracellular matrix macromolecule deposition were evaluated in human fibroblasts cultured on the membranes. The GA-crosslinked Biomed Extend membrane and the not-crosslinked Bio-Gide membrane reduced cell growth and collagen secretion compared with DPPA-crosslinked biomembranes. When Paroguide and Opocrin were compared, better results were obtained with Paroguide. The greatest amount of transforming growth factor beta1, a growth factor involved in extracellular matrix macromolecule accumulation and in tissue regeneration, was produced by cells cultured on Paroguide, with Opocrin second. Our data suggest that the DPPA method is more biocompatible than the GA for crosslinking collagen biomaterials and that membranes made of collagen plus chondroitin sulfate are better than membranes made of pure collagen.
Assuntos
Azidas/metabolismo , Materiais Biocompatíveis/metabolismo , Colágeno/metabolismo , Glutaral/metabolismo , Membranas Artificiais , Fibroblastos/metabolismo , Glicosaminoglicanos/biossíntese , Humanos , Fator de Crescimento Transformador beta/metabolismoRESUMO
When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.